In vitro and in vivo studies have shown that nonmuscle myosin IIs play important roles in cell adhesion, cell migration, and cell division. Three different genes encoding nonmuscle myosin heavy chain II have been identified in vertebrates (Myh9, Myh10 and Myh14: NMHC II-A, II-B and II-C, respectively). To understand the in vivo function of these myosins, we have generated a series of knockout and mutant knock-in mice and mice in which GFP-myosin II-A replaced endogenous myosin II-B. Deletion of NMHC II-A leads to lethality at E6.5 due to defects in cell-cell adhesion. Mice ablated for NMHC II-B die between E14.5 and P0 with severe defects in both the heart and brain. The cardiac defects include a VSD, dextroposition of the aorta and a decreased number of cardiac myocytes. In addition to the defect in cytokinesis, NMHC II-B-ablated cardiac myocytes show abnormalities in karyokinesis. The brain defects include the impaired migration of groups of neuronal cells and the early development of a severe hydrocephalus. The cause of the hydrocephalus appears to be a defect in cell-cell adhesion in the cells lining the embryonic spinal canal, which results in the complete obstruction of the canal by the underlying neuroepithalial cells. Interestingly, when NMHC II-A is knocked-in to replace the endogenous NMHC II-B, defects in cell-cell adhesion and hydrocephalus are rescued, due to the presence of II-A at the adhesion sites. This defect can also be rescued by a mutant form of NMHC II-B, which cannot dissociate from actin.[unreadable] Mutations in both NMHC II-A and II-C have been reported in human patients, but no mutation in NMHC II-B in humans has yet been identified. In order to create a potential model for human diseases, we generated mice carrying a single amino acid mutation in the motor domain of NMHC II-B. The mutant mice show novel phenotypes not observed in B-/B- mice. These include a failure in ventral body wall closure, ectopia cordis, defects in remodeling of the endocardial cushions, and abnormal development of the diaphragm, resembling the human syndrome Pentalogy of Cantrell. These novel defects are consistent with a dominant negative effect of the mutant form of NMHC II-B on other isoforms of nonmuscle myosin. A second model for human disease results from the replacement of NMHC II-B with II-A or the time-delayed cardiac-specific ablation of II-B. In both cases, the mice developed a dilated cardiomyopathy.[unreadable] Our results show that each myosin II plays specific roles during mouse development, but, in some cases, they are interchangeable. More importantly, they indicate that: (1) the myosin II isoforms can act together to fulfill some cellular processes; (2) some cellular processes rely more on the structural function of nonmuscle myosin II, rather than its motor activity.